US11401317B2 - Human-CD123-targeting chimeric receptor ligand and application thereof - Google Patents

Human-CD123-targeting chimeric receptor ligand and application thereof Download PDF

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US11401317B2
US11401317B2 US16/329,739 US201716329739A US11401317B2 US 11401317 B2 US11401317 B2 US 11401317B2 US 201716329739 A US201716329739 A US 201716329739A US 11401317 B2 US11401317 B2 US 11401317B2
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Qiuchuan ZHUANG
Xiaohu Fan
Lei Yang
Pingyan WANG
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Nanjing Legend Biotechnology Co Ltd
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Definitions

  • the present invention belongs to the field of biomedical or biopharmaceutical technologies, and relates to a human CD123-targeting chimeric receptor ligand and an application thereof.
  • CD123 i.e., human interleukin-3 receptor a chain (Interleukin-3 receptor subunit alpha, IL-3R ⁇ ), has a full length of 378 amino acids (NP_002174.1), wherein a signal peptide comprises 18 amino acids (1-18), an extracellular region comprises 287 amino acids (19-305), a transmembrane region comprises 20 amino acids (306-325), and an intracellular region comprises 53 amino acids (326-378).
  • AML acute myeloid leukemia
  • B-ALLs acute B-cell lymphocytic leukemia
  • hairy cell leukemia and blastic plasmacytoid dendritic cell tumor (BPDCN).
  • Mu ⁇ oz L et al. detected 64 acute leukemia patients and found that 43 out of 45 AML patients thereof were CD123 positive, 2 M7 AML patients were CD123 negative, 13 B-cell acute leukemia patients thereof were all CD123 positive, while 6 T-cell acute leukemia patients were all CD123 negative (Haematologica. 2001, December; 86 (12): 1261-9).
  • Human interleukin-3 is a native ligand of CD123.
  • Human IL-3 gene is located on human chromosome V (5q23-31) and has a molecular weight of approximately 15-17 kDa.
  • IL-3 molecule is not conserved in evolution, and there is only 29% amino acid homology between human IL-3 molecules and mouse IL-3 molecules.
  • DT388IL-3 is a fusion protein of IL-3 molecule and diphtheria toxin.
  • DT388IL-3 significantly increased the survival of mice carrying IL-3 receptor positive AML cells with no significant side effects (Leukemia. 2003 January; 17 (1): 155-9).
  • DT388IL-3 In the safety assessment of cynomolgus monkeys, DT388IL-3 also showed good safety (Leuk Lymphoma. 2004, August; 45 (8): 1647-56). The good safety of DT388IL-3 also discloses the safety of use of interactions of ligands and receptors between IL-3 and CD123 for treatment of diseases.
  • CAR-T Chimeric Antigen Receptor Modified T cell therapy
  • Chimeric antigen receptor consists of a tumor-associated antigen-binding region or antigen-binding domain, an extracellular hinge region, a transmembrane region, an intracellular signal transduction domain, etc.
  • the CAR antigen-binding domain typically consists of antibodies in different forms.
  • Most of the developed CAR-T immunotherapy have antigen recognition sequences in a form that is based on scFv antibody fragments.
  • the anti-human CD123 murine antibodies used by relevant research teams in the Abramson Cancer Center at the University of Pennsylvania and in the City of Hope, USA include clone32716 and clone26292, wherein clone32716 exhibits better in vitro and in vivo antitumor activities of killing CD123-positive cells than clone26292 does, and can remove CD123-positive tumor cells in the mouse models within two weeks (J Immunother. 2007, September; 30(6): 607-13; US20140271582A1; Blood. 2013; 122(18): 3138-3148). French CELECTIS has also developed a CD123 CAR-T vector, and the CD123 antibody used therein is Klon43 (WO2015193406A1).
  • Clinical trials of CAR-T therapy for AML registered in the US Clinical Trials registration website mainly include NCT02159495 in the City of Hope and NCT02623582 in the Abramson Cancer Center of the University of Pennsylvania.
  • These CAR-T cells and immunotherapies thereof that are designed based on scFv antibodies all may lead to off-target effects that are common to CAR technologies due to possible cross reactivity among the selected antibodies themselves. Therefore, it is necessary to cautiously verify their safety in clinical trials. Since all of the above CD123 CAR-T use murine monoclonal antibodies as CD123 targeting molecules, a potential immunogenicity may exist when they are applied to a human body.
  • the present invention provides a human CD123-targeting Chimeric Receptor Ligand (CRL), which is different from the conventional CAR configuration based on antibody scFv fragments, but a brand-new, antibody-independent, and human CD123-targeting structure similar to that of CAR.
  • the human CD123-targeting CRL (CD123 CRL) may comprise a native IL-3 molecule-based CD123-specific binding domain, a hinge region or non-hinge region, a transmembrane region, an intracellular signal transduction domain, etc.
  • the present invention further provides a CRL-modified immune cell, i.e., CD123 CRL-modified T cell (CRL-T).
  • the human CD123-targeting CRL and immune cells thereof according to the present invention have CD123 target specific tumor killing effect and safety that are better than those of conventional scFv antibody-based CD123 CAR-T; the use of the native human IL-3 molecule-based CD123-targeting molecules for immunotherapy does not lead to heterologous immunogenicity; and the present invention is very valuable in clinical treatment of CD123 positive blood diseases and other diseases.
  • An object of the present invention is to provide a human CD123-targeting chimeric receptor ligand.
  • Another object of the present invention is to provide a genetically engineered immune effector cell.
  • Another object of the present invention is to provide an application of the human CD123-targeting chimeric receptor ligand and the immune effector cell thereof.
  • a further object of the present invention is to provide a method for preparing human CD123-targeting chimeric receptor ligands and immune effector cells thereof.
  • a human CD123-targeting chimeric receptor ligand comprising an IL-3 molecule-based CD123 specific binding domain, transmembrane region and intracellular signaling domain.
  • the intracellular signaling domain of the human CD123-targeting chimeric receptor ligand comprises a signaling domain and/or co-stimulatory signaling domain.
  • the signaling domain of the intracellular signaling domain can be selected from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12 molecules, preferably CD3 zeta molecules.
  • the co-stimulatory signaling domain of the intracellular signaling domain can be selected from intracellular domains of the following signal molecules: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,
  • the intracellular signaling domain is more preferably selected from intracellular signaling domains of the following molecules: CD3 zeta, 4-1BB and/or CD28.
  • the human CD123-targeting chimeric receptor ligand comprises a protein secretion related signal peptide sequence that can be selected from CD8 ⁇ signal peptide, GM-CSFR ⁇ signal peptide, CD4 signal peptide, or IL-3 signal peptide, preferably IL-3 signal peptide.
  • the extracellular and transmembrane regions of the human CD123-targeting chimeric receptor ligand may comprise a Linker structure or do not comprise a Linker structure.
  • the human CD123-targeting chimeric receptor ligand according to the present invention may comprise two or more repeating CD123 chimeric receptor ligands in serial connection, a plurality of IL-3 molecule based CD123 binding domains in mutual serial connection, or a combination of multivalent target-specific chimeric receptor ligands formed by an IL-3 molecule based CD123 binding domain and an antibody-based antigen binding domain in serial connection, wherein repeating units can be connected by one or more Linker structures therebetween.
  • the human CD123-targeting chimeric receptor ligand is characterized in that the encoded amino acid sequence is shown by SEQ ID NO: 14 to SEQ ID NO: 19, or is a modified amino acid sequence having 85% to 99%, 90% to 99%, or 95% to 99% identity therewith.
  • a nucleic acid molecule is characterized by a nucleotide sequence that encodes the human CD123-targeting chimeric receptor ligand.
  • the nucleic acid molecule is characterized in that the nucleotide coding sequence is shown by SEQ ID NO: 33 to SEQ ID NO: 38.
  • a vector is characterized by comprising the nucleic acid sequence that encodes the human CD123-targeting chimeric receptor ligand.
  • a genetically engineered immune effector cell comprises a gene sequence that encodes the human CD123-targeting chimeric receptor ligand according to the present invention.
  • the immune effector cell is preferably selected from T-lymphocyte cells, NK cells, and immune cells from culturing and differentiation of hematopoietic stem cells, pluripotent stem cells or embryonic stem cells, more preferably T lymphocytes.
  • the genetically engineered immune cells are characterized in that the expressed human CD123-targeting chimeric receptor ligand comprises an IL-3 molecule-based CD123 specific binding domain, a transmembrane region, and an intracellular signaling domain.
  • the genetically engineered immune cells are characterized in that the expressed human CD123-targeting chimeric receptor ligand comprises an amino acid sequence shown by SEQ ID NO: 14, or an amino acid sequence having 85% to 99%, 90% to 99%, or 95% to 99% identity therewith.
  • the genetically engineered immune cells are characterized in that the expressed human CD123-targeting chimeric receptor ligand comprises an amino acid sequence shown by SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, or an amino acid sequence having 85% to 99%, 90% to 99%, or 95% to 99% identity therewith.
  • the present invention provides a method for preparing the above-described human CD123-targeting chimeric receptor ligand and modified immune effector cells thereof.
  • the present invention relates to a use of the human CD123-targeting chimeric receptor ligand and immune effector cells thereof in the manufacture of a medicament for anti-tumor immunotherapy, preferably a use in the manufacture of a medicament for treating acute myeloid leukemia.
  • the present invention provides a human CD123-targeting CRL (CD123 CRL), which creatively uses a human IL-3 molecule as an extracellular specific CD123 molecule-targeting binding region, specifically recognizes tumor-associated surface antigen CD123 through ligand/receptor, then transfers recognition signals into cells through intracellular co-stimulatory molecules, activates the killing effect of immune cells, and thus reduces and eliminates tumor cells.
  • the present invention provides a human CD123-targeting CRL-modified T cell (CD123 CRL-T), which can specifically bind with CD123 on the surface of tumor cells, thereby having a specific killing effect on tumor cells.
  • CD123 CRL-T exhibits better CD123 target specific tumor killing effect and cytokine release amount, thereby improving specificity and safety of targeted treatment.
  • FIGS. 1 a and 1 b show a schematic structural diagram of a human CD123-targeting chimeric receptor ligand constructed according to the present invention.
  • FIG. 1 a is a schematic structural diagram of the IL-3 based CD123 chimeric receptor ligand according to the present invention, wherein the structure of the signal peptide part can be selected from IL-3 and CD8 ⁇ molecules. 4-1BB and/or CD28 signaling molecules are used as examples of the co-stimulatory signaling domain.
  • FIG. 1 b is a schematic structural diagram of conventional scFv antibody-based CD123 CAR-T.
  • FIG. 2 is a schematic diagram of a human CD123-targeting chimeric receptor ligand vector constructed according to the present invention.
  • the structural diagram of the CD123 CRL vector constructed according to the present invention is illustrated with CRL1 as an example, and the illustrated CRL1 comprises IL-3 extracellular signal peptide, CD123 binding domain, CD8 ⁇ hinge region (Hinge) and transmembrane region (TM), the 4-1 BB intracellular co-stimulatory domain (Cyto) and CD3z signaling domain.
  • FIG. 3 illustrates verification of K562.CD123.Luc cell lines.
  • CD123 molecules in the constructed clone 8 cell lines have the highest expression level, Luciferase also has a high expression level, and clone 8 is named as K562.CD123.Luc.
  • FIGS. 4 a and 4 b illustrate a study on in vitro active functions of the CD123-targeting chimeric receptor ligand.
  • the Y-axis shows relatively activity RLU of the remaining luciferase in reaction wells and relative amount of living cells in the corresponding wells when co-culture ends.
  • CAR1-T, CAR2-T, CAR3-T and CRL1-T cells all have good in vitro cytotoxicity against RPMI8226.Luc cells.
  • CAR1-T, CAR2-T, CAR3-T and CRL1-T cells can all release cytokines IFN ⁇ in the presence of RPMI8226.Luc cells. In the absence of RPMI8226.Luc cells, on the other hand, CRL1-T exhibits a lower release amount of IFN ⁇ cytokine than those by CAR1-T, CAR2-T and CAR3-T.
  • FIGS. 5 a -5 c illustrate a detection test on cytokine IFN- ⁇ release level of IL-3 molecule based CD123 CRL-T.
  • FIG. 5 a illustrates the IFN- ⁇ level released by CRL1-T cells in the presence of K562.CD123.Luc that causes over expression and low expression of CD123 molecules;
  • FIG. 5 b illustrates the IFN- ⁇ level released by CRL1-T cells under stimulation by RPMI8226.Luc cells that cause low expression of CD123 molecules;
  • FIG. 5 c illustrates the IFN- ⁇ level released by CRL1-T cells under stimulation by CD123-negative A549.Luc cells.
  • FIGS. 6 a -6 c illustrate a detection test on cytokine IL-2 release level of IL-3 molecule based CD123 CRL-T.
  • FIG. 6 a illustrates the IL-2 level released by CRL1-T cells in the presence of K562.CD123.Luc that causes over expression and low expression of CD123 molecules;
  • FIG. 6 b illustrates the IL-2 level released by CRL1-T cells under stimulation by RPMI8226.Luc cells that cause low expression of CD123 molecules;
  • FIG. 6 c illustrates the IL-2 level released by CRL1-T cells under stimulation by CD123-negative A549.Luc cells.
  • FIGS. 7 a -7 d illustrate assessment of functionality and specificity of the IL-3 based CD123 CRL-T.
  • FIG. 7 a illustrates that, compared with the negative control UnT cells, CRL1-T has good in vitro cytotoxicity on K562.CD123.Luc cells that cause over expression of CD123;
  • FIG. 7 b illustrates that CRL1-T almost has no in vitro cytotoxicity on CD123-negative K562.CD19.Luc cells;
  • FIG. 7 c illustrates that CRL1-T cells have good in vitro cytotoxicity on RPMI8226.Luc cell lines that cause low expression of CD123;
  • FIG. 7 d illustrates that CRL1-T cells have no in vitro cytotoxicity at all on lung cancer cell line A549.Luc that does not express CD123 molecules.
  • the present invention provides a human CD123-targeting chimeric receptor ligand, comprising an IL-3 molecule-based CD123 binding domain, a transmembrane region, and an intracellular signaling domain.
  • IL-3 molecule-based CD123 binding domain refers to a CD123 binding domain from a native human IL-3 amino acid sequence.
  • the amino acid sequence of native human IL-3 is shown by SEQ ID NO: 40, which contains a total of 152 amino acid residues.
  • the 19 amino acid residues on the N terminal of native human IL-3 molecule are an IL-3 signal peptide, which has an amino acid sequence as shown by SEQ ID NO: 3.
  • SEQ ID NO: 3 If the signal peptide portion (SEQ ID NO: 3) is removed from the native human IL-3 molecule, the SEQ ID NO: 1 sequence (comprising 133 amino acids) can be obtained. SEQ ID NO: 1 can be used as an IL-3 receptor binding domain (also referred to as CD123 binding domain), which has the ability to specifically bind CD123.
  • IL-3 receptor binding domain also referred to as CD123 binding domain
  • SEQ ID NO: 2 (containing 124 amino acids) can be obtained.
  • An IL-3 fragment containing only SEQ ID NO: 2 sequence can also bind CD123, and may be used as an IL-3 receptor binding domain.
  • the “IL-3 molecule-based CD123 binding domain” used herein has the ability to specifically bind CD123.
  • the IL-3 molecule-based CD123 binding domain may comprise at least 100 contiguous amino acid residues of SEQ ID NO: 1, such as 102, 105, 110, 115, or 120 contiguous amino acid residues.
  • the IL-3 molecule-based CD123 binding domain may be SEQ ID NO: 1 or SEQ ID NO: 2.
  • the human CD123-targeting chimeric receptor ligand according to the present invention can comprise one IL-3 molecule-based CD123 binding domain, or can comprise two or more IL-3 molecule-based CD123 binding domains. These CD123 binding domains may or may not be identical. For example, there can be two or more repeating CD123 chimeric receptor ligands in serial connection, a plurality of IL-3 molecule-based CD123 binding domains in mutual serial connection, or a combination of multivalent target-specific chimeric receptor ligands formed by an IL-3 molecule based CD123 binding domain and an antibody-based antigen binding domain in serial connection, wherein repeating units can be connected by one or more Linker structures therebetween.
  • Linker also referred to as the connecting peptide or linker, is a flexible amino acid sequence for connection of two domains. It would be easy for a person skilled in the art to select and prepare a connecting peptide.
  • transmembrane region (TM in short) can be used interchangeably with “transmembrane domain” and refers to a thermodynamically stable protein structure area anchored in the cell membrane.
  • the transmembrane region may be obtained from native proteins and selected from CD8 ⁇ molecule or CD28 molecule. For example, an amino acid sequence shown by SEQ ID NO: 6 or 7.
  • intracellular signaling domain refers to a protein structure area capable of conducting cell effector function signals and directing cells to perform a specific function.
  • the intracellular signaling domain may comprise a signaling domain and/or co-stimulatory signaling domain.
  • identity of amino acid sequences, can be used interchangeably with “sameness” and refers to a degree of similarity between the amino acid sequences determined by sequence comparison software, such as BLAST. Methods and software for comparison of amino acid sequences are well known to a person skilled in the art.
  • a modified amino acid sequences can be obtained by performing substitution, deletion and/or addition of one or several (e.g. 1-15, e.g. 2, 3, 5, 8, 10, or 12) amino acid residues on a known amino acid sequence.
  • the CD123 binding domain shown by SEQ ID NO: 1 in the present invention is modified through a conventional protein engineering technique (e.g. conservative amino acid substitution and the like) to obtain a variant sequence having at least 85% (e.g., 85% to 99%, 90% to 99%, or 95% to 99%) sequence identity with SEQ ID NO: 1, and having substantially the same CD123 binding capacity (e.g., retaining at least 80%, such as at least 85%, 90% or even 95% binding capacity)
  • scFv or “scFv antibody,” refers to a single chain antibody (scFv), which is formed by connecting a heavy chain variable region and a light chain variable region of the antibody via a short peptide having 15 to 20 amino acids.
  • the human CD123-targeting chimeric receptor ligand according to the present invention can further comprise an extracellular signal peptide structure, for example, IL-3 signal peptide or CD8 ⁇ signal peptide.
  • an extracellular signal peptide structure for example, IL-3 signal peptide or CD8 ⁇ signal peptide.
  • the present invention is designed through combining the native human IL-3 molecule and chimeric receptor binding ligand structures, such as co-stimulatory factor.
  • the present invention will be further described below with reference to specific examples.
  • the present invention obtains a nucleotide sequence that encodes the human CD123 molecule through gene synthesis, and GenScript Biotech Corp. in Nanjing provided the gene synthesis service. Under the action of T4 ligase, the synthesized CD123 nucleotide sequence is connected, at 20° C. overnight, to a pLVX-Puro (Clontech, Catalog #632164) lentiviral vector that has been subjected to digestion of BamH1 and XbaI restriction sites in advance. The ligation product is transformed into DH5 ⁇ competent cells and spread onto a bacterial plate. A plurality of clone patches is picked for plasmid extraction (Qiagen Endofree Mega kit). Upon restriction enzyme digestion, sequencing and comparison, sequencing match, the successfully constructed vector is named pLVX-CD123-Puro.
  • the extracted pLVX-CD123-Puro expression plasmid and pCMV- ⁇ R-8.74 and pMD2.G helper plasmids are mixed according to a certain ratio, and 293FT cells are co-transfected.
  • the virus-containing cell culture supernatant is collected and centrifuged for 5 min at 4° C. and 3000 rpm. After filtration through a 0.45 ⁇ m filter, the supernatant is subjected to high speed centrifugation for 120 min at 4° C. and 25000 rpm. The supernatant is discarded, a concentrated virus solution is obtained through resuspension and dissolution, and the concentrated virus solution is stored at ⁇ 80° C. for later use.
  • a lentiviral vector carrying the firefly luciferase/Luciferase reporter gene is prepared also by the same method.
  • the K562 cell line is purchased from ATCC (Catalog #CCL-243), and is cultured conventionally using 90% IMDM (Life technology, Catalog #A10491-01)+10% FBS (Life technology, Catalog #10099-141)+1% penicillin/streptomycin (Life technology, Catalog #15140-122).
  • the constructed lentiviral vector carrying CD123 genes and the lentiviral vector carrying Firefly luciferase/Luciferase reporter gene are added to the culture supernatant of K562 cells for co-transduction into K562 cells.
  • CD123 expression identification is performed using flow cytometry on monoclonal cells obtained above. K562 cells that are not transduced with the CD123 gene are used as negative control. The cell suspension having a cell number of 5 ⁇ 10 5 is pipetted to a 15 ml centrifuge tube for centrifugation at room temperature and 300 g, and the supernatant is discarded after 10 min DPBS is used for resuspension and washing once, and then it is re-suspended in 200 ⁇ l DPBS.
  • 2 ⁇ l anti-CD123-FITC antibody (Miltenyi, Catalog #130-098-886) is further added for incubation at room temperature for 45 min 1 ml DPBS is added, centrifugation is performed at room temperature and 300 g, and the supernatant is discarded after 10 min 1 ml DPBS is further added, centrifugation is performed at room temperature and 300 g, and the supernatant is discarded after 10 min Lastly, 200 ⁇ l DPBS-resuspended cells are added, and FITC signals are detected on a FACScalibur flow cytometer (BD Inc.).
  • Luciferase assay is performed using the One-Glo Luciferase assay kit (Promega, Catalog #E6110): the cell density of each clone is adjusted to 2000 cells/20 ⁇ l, which is then added to a 384-well microwell plate, 20 ⁇ l One-Glo Luciferase assay reagent is added to each well, centrifugation is performed at room temperature and 300 g, and after 1 min, the plate is left undisturbed for 10 min Subsequently, chemiluminescence signals are detected on a PHERAstar microplate reader (purchased from BMG).
  • PHERAstar microplate reader purchased from BMG
  • clone 8 also has a relatively high level of Luciferase, and its signal value is 659 times of that of the negative K562 cells control with no transduction (relative enzyme activity 761623/1156).
  • clone 8 is a good cell line, and clone 8 is named K562.CD123.Luc and used as a tool cell in subsequent experiments.
  • codon optimization of a nucleotide sequence of a human IL-3 molecule is performed, so as to optimize the expression in human cells.
  • the codon optimization is implemented using the OptimumGeneTM codon optimization technique of GenScript Biotech Corp. in Nanjing.
  • the IL-3 nucleotide sequence after the codon optimization is shown by SEQ ID NO: 39, the amino acid sequence encoded thereby is shown by SEQ ID NO: 40, and the IL-3 molecule expressed by the gene comprises IL-3 signal peptide and IL-3 receptor binding domain.
  • the CD123 CRL fusion gene fragment is designed in the following order of encoding genes: an extracellular signal peptide, an IL-3-based CD123 binding domain, a hinge region (Hinge), a transmembrane region (TM), an intracellular co-stimulatory signaling domain, and an intracellular signaling domain.
  • GenScript Biotech Corp. in Nanjing provides technical services for synthesis of the fusion gene.
  • the basic structure of the IL-3-based human CD123-targeting chimeric receptor ligand according to the present invention is shown in Table 2, comprising a chimeric receptor ligand structure shown by CRL1 to CRL6, and the corresponding amino acid sequences of the chimeric receptor ligand thereof are shown by SEQ ID NO: 14 to SEQ ID NO: 19, respectively.
  • conventional CARs are also designed as experimental controls, and the fusion gene is directly synthesized using a gene synthesis technique in an order of CD8 ⁇ signal peptide, anti-CD123 scFv, CD8 ⁇ hinge region (Hinge), CD8 ⁇ transmembrane region (TM), 4-1BB intracellular co-stimulatory signaling domain, and CD3 ⁇ intracellular signaling domain, respectively, as the CAR1, CAR2 and CAR3 structures shown in Table 2.
  • CD123 CRL The major structural components and sequence of CD123 CRL designed by the present invention is as follows:
  • CD123 binding domain comprises at least 100 contiguous amino acid sequences in the amino acid sequence shown by SEQ ID NO: 1.
  • the IL-3 molecule-based CD123 binding domain comprises the amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO: 2.
  • the nucleotide encoding sequence thereof is shown by SEQ ID NO: 20 or SEQ ID NO: 21.
  • Extracellular signal peptide it may be selected from IL-3 signal peptide or CD8 ⁇ signal peptide.
  • the amino acid sequence of the IL-3 signal peptide is shown by SEQ ID NO: 3, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 22; the amino acid sequence of the CD8 ⁇ signal peptide is shown by SEQ ID NO: 4, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 23.
  • Hinge region it may be selected from hinge regions of a variety of molecules, the CD8 ⁇ hinge region is selected for CRL1, the amino acid sequence thereof is shown by SEQ ID NO: 5, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 24.
  • Transmembrane domain it may be selected from CD8 ⁇ transmembrane region or CD28 transmembrane region.
  • the amino acid sequence of the CD8 ⁇ transmembrane region is shown by SEQ ID NO: 6, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 25; the amino acid sequence of the CD28 transmembrane region is shown by SEQ ID NO: 7, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 26.
  • Intracellular signaling domain the amino acid sequence of CD3 zeta is shown by SEQ ID NO: 8, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 27.
  • Intracellular co-stimulatory signaling domain selected from 4-1BB signaling molecule or CD28 signaling molecule.
  • the amino acid sequence of the 4-1BB signaling molecule s is shown by SEQ ID NO: 9, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 28.
  • the amino acid sequence of the CD28 signaling molecule is shown by SEQ ID NO: 10, and the nucleotide encoding sequence thereof is shown by SEQ ID NO: 29.
  • the pLVX-Puro vector purchased from Clontech is subjected to a digestion reaction with ClaI and EcoRI restriction enzymes to knock out the CMV promoter, and the human EF1 ⁇ promoter (GenBank: J04617.1) is cloned into the digested vector to obtain a pLVX-hEF1 ⁇ vector.
  • the CD123 CRL fusion gene sequence from gene synthesis is cloned to an expression plasmid pLVX-hEF1 ⁇ to form a recombinant CRL expression plasmid.
  • the recombinant CRL expression plasmid (pLLV-CRL) is extracted and mixed with pCMV-AR-8.74 and pMD2.G helper plasmids according to a certain ratio, and 293FT cells are co-transfected.
  • the virus-containing cell culture supernatant is collected and centrifuged for 5 min at 4° C. and 3000 rpm. After filtration through a 0.45 ⁇ m filter, the supernatant is subjected to high speed centrifugation for 120 min at 4° C. and 25000 rpm. The supernatant is discarded, a concentrated virus solution is obtained through resuspension and dissolution, and the concentrated virus solution is stored at ⁇ 80° C. for later use.
  • Recombinant CRL lentivirus vectors obtained using the above method are named as pLLV-CRL1, pLLV-CRL2, pLLV-CRL3, . . . , pLLV-CRL6, respectively.
  • scFv antibody sequences of the above-described clone 32716, clone 26292, and Klon43 are synthesized and cloned into the lentiviral vector according to the present invention, which are named as pLLV-CAR1, pLLV-CAR2 and pLLV-CAR3, respectively.
  • the construction of the above vectors employs conventional molecular biology techniques, i.e., digestion, ligation, transformation, and cloning identification techniques, which can be easily grasped and operated by those of ordinary skills in the art.
  • peripheral blood mononuclear cells 50 mL of fresh peripheral blood is taken from volunteers, and peripheral blood mononuclear cells (PBMC) are isolated using lymphocyte isolation liquid and density gradient centrifugation method.
  • PBMC peripheral blood mononuclear cells
  • a Pan T Cell Isolation Kit (Miltenyi Biotech) is used to label the cells with magnetic beads, and T lymphocytes are isolated and purified.
  • CD3/CD28 magnetic beads are further used to perform T lymphocyte activation and proliferation on the purified T cells.
  • Activated T lymphocytes are collected and resuspended in RPMI1640 medium.
  • Lentivirus is used infect 1 ⁇ 10 6 activated T lymphocytes, the cell suspension is added to 6-well plates, which are placed in an incubator at 37° C. and 5% CO 2 overnight. On the second day, centrifugation is performed again, and the culture is replaced with a fresh medium. Fresh medium is added every 2 days to continue expanding the culture.
  • the prepared CRL-T and CAR-T cells are collected through centrifugation, the cells are washed 3 times with DPBS, and then genomic DNA is prepared using a human genome extraction kit Gentra Puregene Cell Kit (purchased from Qiagen).
  • Gentra Puregene Cell Kit purchased from Qiagen.
  • the OD 260 nm and OD 280 nm absorbance of the prepared DNA is detected using NanoDrop2000 (Thermo Scientific), and the concentration is calculated.
  • 50 ⁇ l reaction system is configured according to the instructions for the kit SYBR Green Realtime PCR Master mix plus (purchased from Toyobo), and then the gene copy number is detected on a fluorescence quantitative PCR instrument (ABI #7300).
  • the qPCR detection uses a plasmid containing a fragment of interest that has been accurately quantified as a positive control, and a standard curve is plotted. Straight lines are plotted according to the CT values of qPCR at various copy number concentrations and corresponding copy numbers to fit the standard curve. Relative copy numbers are calculated for other detection samples according to the fitting equation of the standard curve.
  • CAR expressed by CAR-T cells is used as a positive control, and non-transduced T-lymphocytes (UnT) are used as a blank control.
  • UnT non-transduced T-lymphocytes
  • Detection results of copy numbers of CRL and CAR integration are shown in Table 3.
  • the results show that CRL1 gene integration is detected in the CRL1-T cell genome, and its copy number (2.34 ⁇ 10 5 copies/ng genomic DNA) is equivalent to the copy number of CAR gene transduced in CAR-T cells (3.1 ⁇ 10 4 to 2.77 ⁇ 10 5 copies/ng genomic DNA), and the UnT detection value is very low (16 copies/ng genomic DNA) for the blank control, which is the detection background.
  • RPMI8226 cells express a certain level of CD123 receptors (proteinatlas.org/ENSG0000018 5291-IL-3RA/tissue), and it has been reported that CD123 molecule-targeting CAR-T has good in vitro cytotoxicity on RPMI8226 cells (WO2015193406A1).
  • a RPMI8226 cell line that stably expresses Luciferase is constructed (RPMI8226.Luc), and Example 1 can be referenced to for the construction method.
  • RPMI8226.Luc cells are used as model cells for the study on in vitro functions of CRL-T cells, and CAR1, CAR2, CAR3 modified T cells are used as controls, which are named as CAR1-T, CAR2-T, and CAR3-T, respectively.
  • CRL-T cells, CAR-T cells, and UnT cells are co-cultured overnight with target cells, respectively, at a ratio of 20:1 at 37° C. and 5% CO 2 .
  • target cells respectively, at a ratio of 20:1 at 37° C. and 5% CO 2 .
  • the ONE-GloTM Luciferase Assay reagent is added.
  • a PHERAStar Plus is used to detect RLU reading and evaluate in vitro cytotoxicity.
  • the Y-axis shows the remaining relative luciferase activity (RLU, relative light unit) in reaction wells after the end of co-culture.
  • the displayed luciferase RLU value is high, it indicates that there are many target cells remaining in the reaction wells that are not killed and the cytotoxicity in the wells is low; conversely, if the displayed luciferase RLU value is low, it indicates that there are not many target cells remaining in the reaction wells that are not killed and the cytotoxicity in the wells is high.
  • CAR1-T, CAR2-T, CAR3-T, and CRL1-T cells all have good in vitro cytotoxic activities on RPMI8226.Luc cells, wherein the relative Luciferase signal value of the remaining viable cells in the CAR1-T treated group is 1413 ⁇ 152, the relative Luciferase signal value of the remaining viable cells in the CAR2-T treated group is 3883 ⁇ 423 RLU, the relative Luciferase signal value of the remaining viable cells in the CAR3-T treated group is 3184 ⁇ 262, and the relative Luciferase signal value of the remaining viable cells in the CRL1-T treated group is 4531 ⁇ 276, which show good killing activities relative to the relative Luciferase signal value of the remaining viable cells in the UnT treated group that is 9442 ⁇ 1553.
  • cytokines ⁇ -interferon/IFN- ⁇ and interleukin-2/IL-2
  • a good CAR-T vector can release the above cytokines in the presence of a target antigen, while the release level is relatively low in the absence of a target antigen.
  • the detection results in FIG. 4 b show that CAR1-T, CAR2-T, CARS-T, and CRL1-T cells can all release the cytokine IFN ⁇ in the presence of RPMI8226.Luc cells.
  • CAR1-T has better in vitro cytotoxicity than that of CAR2-T, CARS-T, and CRL1-T ( FIG. 4 a ), but CAR1-T can release, in the absence of RPMI8226.Luc target cells, the cytokine IFN ⁇ at a level that is significantly higher than that of the cytokine IFN ⁇ released by CAR2-T, CARS-T, and CRL1-T, indicating a relatively high non-specificity.
  • CRL1-T shows a release amount of the cytokine IFN ⁇ that is lower than that of CAR1-T, CAR2-T, and CARS-T, and at the same time, CRL1-T has a cytotoxicity on RPMI8226.Luc that is equivalent to that of CAR2-T and CARS-T, indicating a relatively good in vitro effectiveness and safety.
  • the CRL1 vector is preferably selected for T cell modification, and stimulation tests by cytokines IFN- ⁇ and IL-2 are performed on CD123-positive tumor cells.
  • CRL-T cells and different target cells are co-cultured overnight at certain ratios, 37° C. and 5% CO 2 . After the co-culture is ended, the test microplates are centrifuged at 200 g for 5 min, and then a part of the supernatant is carefully taken out for detection of IFN- ⁇ and IL-2 secretion levels in the supernatant using a kit for real-time fluorescence resolution technique (HTRF, Cisbio #64IL2PEB).
  • HTRF real-time fluorescence resolution technique
  • CRL1-T cells release a high level of IFN- ⁇ (4383 ⁇ 236.1 pg/ml), whereas negative T cells (UnT) have a very low level of release (108.6 ⁇ 23.97 pg/ml) that is close to the baseline (29.78 ⁇ 37.52 pg/ml); when K562.CD123.Luc is missing, CRL1-T cells release a very low level of background IFN- ⁇ (109.3 ⁇ 10.41 pg/ml) that is close to the baseline (29.78 ⁇ 37.52 pg/ml).
  • CRL1-T cells can also release a higher level of IFN- ⁇ (697.4 ⁇ 0.00 pg/ml, 261.6 ⁇ 3.1 pg/ml) than UnT cells do ( FIG. 5 b ).
  • CRL1-T cells are similar to the UnT cell control, both of which release a low level of IFN- ⁇ (315 ⁇ 0.00 pg/ml, 58.54 ⁇ 11.53 pg/ml) ( FIG. 5 c ).
  • CRL1-T cells release a high level of IL-2 (2.41 ⁇ 0.06 IU/ml), whereas UnT cells have a very low level of release (0.60 ⁇ 0.00 IU/ml) that is close to the baseline (0.59 ⁇ 0.00 IU/ml); when there is no stimulation by K562.CD123.Luc cells, CRL1-T cells release a very low level of IL-2 (0.59 ⁇ 0.00 IU/ml) that is the same as the baseline (0.59 ⁇ 0.00 IU/ml).
  • CRL1-T cells As stimulated by RPMI8226.Luc cells having a low expression of CD123 molecules, CRL1-T cells also release a relatively low level of IL-2 (0.61 ⁇ 0.00 IU/ml) ( FIG. 6 b ). As stimulated by CD123-negative A549.Luc cells, CRL1-T cells are similar to the UnT cell negative control, both of which release a very low level of IL-2 (0.60 ⁇ 0.00 IU/ml, 0.60 ⁇ 0.00 IU/ml) ( FIG. 6 c ).
  • CRL-T cells, CAR-T cells, and UnT cells are co-cultured overnight with target cells, respectively, at a ratio of 20:1 at 37° C. and 5% CO 2 .
  • target cells respectively, at a ratio of 20:1 at 37° C. and 5% CO 2 .
  • the ONE-GloTM Luciferase Assay reagent is added.
  • a PHERAStar Plus is used to detect RLU reading and evaluate in vitro cytotoxicity.
  • FIG. 7 just like in Example 4, the Y-axis shows the remaining relative luciferase activity (RLU) in reaction wells after the end of co-culture, which corresponds to the relative number of viable cells in the wells.
  • the displayed luciferase RLU value is high, it indicates that there are many target cells remaining in the reaction wells that are not killed and the cytotoxicity in the wells is low; conversely, if the displayed luciferase RLU value is low, it indicates that there are not many target cells remaining in the reaction wells that are not killed and the cytotoxicity in the wells is high.
  • CRL1-T shows a good in vitro cytotoxic effect on K562.CD123.Luc cells that overexpress CD123 with 97.5% of K562.CD123.Luc cells killed by CRL1-T and without being killed by the negative control UnT;
  • the Luciferase relative light value of the remaining cells in the CRL1 killing group is 7307 ⁇ 3639, whereas the Luciferase relative light value of the remaining cells in the negative control UnT cell killing group is 292420 ⁇ 19102 ( FIG. 7 a ).
  • CRL1-T has almost no in vitro cytotoxicity on CD123-negative K562.CD19.Luc cells; the Luciferase relative light value of the remaining cells in the CRL1 killing group is 411629 ⁇ 14399, whereas the Luciferase relative light value of the remaining cells in the negative control UnT cell killing group is 458030 ⁇ 11222 ( FIG. 7 b ).
  • FIG. 7 a and FIG. 7 b show that the cytotoxicity of CRL1-T has strict target specificity.
  • in vitro killing experiments are further performed on the multiple myeloma cell line RPMI8226.Luc having a low expression of CD123 molecules and the lung cancer cell line A549.Luc that does not express CD123 molecules. As shown in FIG.
  • CRL1-T cells have a good in vitro cytotoxic effect on the RPMI8226.Luc cell line: the Luciferase relative light value of the remaining cells in the CRL1 killing group is 2467 ⁇ 239.6, whereas the Luciferase relative light value of the remaining cells in the negative control UnT cell killing group is 13248 ⁇ 331.3; while CRL1-T has no in vitro cytotoxicity at all on the lung cancer cell line A549.Luc that does not express CD123 molecules: the Luciferase relative light value of the remaining cells in the CRL1 killing group is 577341 ⁇ 17365, whereas the Luciferase relative light value of the remaining cells in the negative control UnT cell killing group is 522895 ⁇ 14198 ( FIG. 7 d ).
  • FIG. 7 c and FIG. 7 d further show that the cytotoxicity of CRL1-T cells has strict target specificity and safety.
  • Amino acid sequence of IL-3 receptor binding domain SEQ ID NO: 1 1-133aa APMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVK SLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF
  • Amino acid sequence of IL-3 receptor binding domain SEQ ID NO: 2 1-124aa APMTQTTPLKTSWVNCSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVK SLQNASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQ Amino acid sequence of IL-3 signal

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